This project investigates the mechanism of sex-specific gene regulation of steroid hydroxylases (P-450s) in mouse liver and kidney. Type I P-45015 alpha is female-specific in liver but male- specific in kidney. Type II P-45015 alpha on the other hand, is female-specific in both tissues. We characterized genes 15 alpha 0H-1 and -2 (-8 Kbp), which encode Type I and II P-45015 alpha, respectively. Despite the differences in sex-specific expressions, both gene structures are nearly identical; there is 96% homology including their flanking and intronic sequences. Although the TATA and CAAT boxes and GRE are located identical positions in the 5'- flanking regions, GC-box-like motif and SV40 enhancer are specific to 15 alpha 0H-1 and -2, respectively. The consensus provides a potential sequence by which the genes are differentially regulated by growth hormone and androgen. There are only 11 substitutions in the total 494 amino acid residues between Type I and II P-45015 alpha. We expressed both types of P-45015 alpha in COS-1 cells and discovered that Type I is the major steroid 15 alpha-hydroxylase, while Type II is the coumarine 7-hydroxylase. We characterized five highly homologous genes, ca, cb, cc, cd, and ce within the mouse male-specific steroid 16 alpha-hydroxylase gene family. Gene ca encode the male-specific 16 alpha-hydroxylase (C-P-4516 alpha) whose expression is regulated by growth hormone and androgen in liver and kidney. Gene cb, not sex-specific, is expressed only in liver. The gene-encoded P-450 whose protein sequence homology to the C-P-45016 alpha is 85%, possesses no steroid 16 alpha- hydroxylase activity. The remaining three genes' expressions are not detected in either tissues. There are several divergent areas in the 5'-flanking regions of all five genes, by which the differential gene expressions might be regulated. We determined that the female-specific I-P-45016 alpha gene is -40 Kbp and is a member of a large multigene family consisting of at least 16 genes. We now are investigating the regulation mechanism of this gene by Rip or Ripr locus on mouse chromosome 7.